Genome editing of FTR42 improves zebrafish survival against virus infection by enhancing IFN immunity.

The development of CRISPR-Cas9 technology introduces an efficient tool for precise engineering of fish genomes. With a short reproduction cycle, zebrafish infection mode can be referenced as antiviral breeding researches in aquaculture fish. Previously we identified a crucian carp-specific gene ftrca1 as an inhibitor of interferon response in vitro. Here, we demonstrate that genome editing of zebrafish ftr42, a homolog of ftrca1, generates a zebrafish mutant (ftr42lof/lof) with an improved resistance to SVCV infection. Zebrafish ftr42 acts as a virus-induced E3 ligase and downregulates IFN antiviral response by facilitating TBK1 protein degradation and also IRF7 mRNA decay. Genome editing results in loss of function of zebrafish ftr42, which enables zebrafish to have enhanced interferon response, thus improving zebrafish survival against virus infection. Our results suggest that fine-tuning fish IFN innate immunity through genome editing of negative regulators can genetically improve viral resistance in fish.

A zebrafish NLRX1 isoform downregulates fish IFN responses by targeting the adaptor STING.

In mammals, NLRX1 is a unique member of the nucleotide-binding domain and leucine-rich repeat (NLR) family showing an ability to negatively regulate IFN antiviral immunity. Intron-containing genes, including NLRX1, have more than one transcript due to alternative splicing; however, little is known about the function of its splicing variants. Here, we identified a transcript variant of NLRX1 in zebrafish (Danio rerio), termed NLRX1-tv4, as a negative regulator of fish IFN response. Zebrafish NLRX1-tv4 was slightly induced by viral infection, with an expression pattern similar to the full-length NLRX1. Despite the lack of an N-terminal domain that exists in the full-length NLRX1, overexpression of NLRX1-tv4 still impaired fish IFN antiviral response and promoted viral replication in fish cells, similar to the full-length NLRX1. Mechanistically, NLRX1-tv4 targeted STING for proteasome-dependent protein degradation by recruiting an E3 ubiquitin ligase RNF5 to drive the K48-linked ubiquitination, eventually downregulating the IFN antiviral response. Mapping of NLRX1-tv4 domains showed that its N-terminal and C-terminal regions exhibited a similar potential to inhibit STING-mediated IFN antiviral response. Our findings reveal that like the full-length NLRX1, zebrafish NLRX-tv4 functions as an inhibitor to shape fish IFN antiviral response.IMPORTANCEIn this study, we demonstrate that a transcript variant of zebrafish NLRX1, termed NLRX1-tv4, downregulates fish IFN response and promotes virus replication by targeting STING for protein degradation and impairing the interaction of STING and TBK1 and that its N- and C-terminus exhibit a similar inhibitory potential. Our results are helpful in clarifying the current contradictory understanding of structure and function of vertebrate NLRX1s.

Zebrafish HERC7c Acts as an Inhibitor of Fish IFN Response.

In humans, four small HERCs (HERC3-6) exhibit differential degrees of antiviral activity toward HIV-1. Recently we revealed a novel member HERC7 of small HERCs exclusively in non-mammalian vertebrates and varied copies of herc7 genes in distinct fish species, raising a question of what is the exact role for a certain fish herc7 gene. Here, a total of four herc7 genes (named HERC7a-d sequentially) are identified in the zebrafish genome. They are transcriptionally induced by a viral infection, and detailed promoter analyses indicate that zebrafish herc7c is a typical interferon (IFN)-stimulated gene. Overexpression of zebrafish HERC7c promotes SVCV (spring viremia of carp virus) replication in fish cells and concomitantly downregulates cellular IFN response. Mechanistically, zebrafish HERC7c targets STING, MAVS, and IRF7 for protein degradation, thus impairing cellular IFN response. Whereas the recently-identified crucian carp HERC7 has an E3 ligase activity for the conjugation of both ubiquitin and ISG15, zebrafish HERC7c only displays the potential to transfer ubiquitin. Considering the necessity for timely regulation of IFN expression during viral infection, these results together suggest that zebrafish HERC7c is a negative regulator of fish IFN antiviral response.

Yellow catfish RIO kinases (RIOKs) negatively regulate fish interferon-mediated antiviral response.

In mammals, right open reading frame kinases (RIOKs) are initially reported to participate in cancer cell proliferation, apoptosis, migration and invasion, and recently they have been related to host immune response. Little is known about the homologs of RIOKs in fish. In the current study, we cloned three homologous genes of RIOK family in yellow catfish (Pelteobagrus fulvidraco), termed Pfriok1, Pfriok2 and Pfriok3. Pfriok1, Pfriok2 and Pfriok3 were constitutively expressed at relatively high levels in yellow catfish tissues, and their mRNA levels were not changed under viral infection. Individual overexpression of PfRIOK1, PfRIOK2 and PfRIOK3 attenuated fish interferon (IFN) response, thereby promoting viral replication in fish cells. Mechanistically, yellow catfish RIOK proteins downregulated fish IFN response through attenuating TBK1 protein levels in cytoplasm. Our findings suggest that yellow catfish RIOK1, RIOK2 and RIOK3 are involved in downregulating fish IFN antiviral response.

Zebrafish MARCH8 downregulates fish IFN response by targeting MITA and TBK1 for protein degradation.

Recent studies have related the membrane-associated RING-CH-type finger (MARCH) family proteins to host innate immune response. Zebrafish (Danio rerio) MARCH8 is reported to target SVCV glycoprotein for degradation; however, little is known about whether fish MARCH8 is involved in innate interferon (IFN) response. In this study, zebrafish march8 was significantly induced by SVCV infection. Overexpression of MARCH8 diminished fish IFN-mediated antiviral response, thus promoting the replication of SVCV and GCRV in fish cells. Mechanistically, MARCH8 interacts with and degrades MITA and TBK1 proteins to inhibit IFN response. Moreover, MARCH8 has an E3 ligase activity and enhances MITA and TBK1 polyubiquitination. Our findings reveal a mechanism whereby zebrafish MARCH8 downregulates fish IFN response and facilitates viral replication by targeting MITA and TBK1 for protein degradation.

Promoter Binding and Nuclear Retention Features of Zebrafish IRF Family Members in IFN Response.

Interferon regulatory factors (IRFs) constitute a family of transcription factors that synchronize interferon (IFN) antiviral response through translocating to nucleus and binding to the promoters of IFN and IFN-stimulated genes (ISGs). Fish contain 11 IRF members; however, whether or how fish IRF family genes function in IFN response remains limited. Herein, we determine the regulatory roles of 11 zebrafish IRF family members in IFN response relevant to their subcellular localization and promoter binding. Zebrafish IRF family members display three patterns of constitutive localization, only in nucleus (IRF1/2/9/11), only in cytoplasm (IRF3/5/7), and largely in nucleus with small amounts in cytoplasm (IRF4b/6/8/10). DNA pull-down assays confirm that all zebrafish IRF proteins are capable to bind fish IFN promoters, albeit to various degrees, thus regulating IFN gene transcription as activators (IRF1/3/5/6/7/8/9/11) or repressors (IRF2/4b/10). Further characterization of distinct IFN gene activation reveals that IRF1/3/5/6/7/8/9/11 efficiently stimulate zebrafish IFNφ1 expression, and IRF1/7/11 are responsible for zebrafish IFNφ3 expression. Two conserved basic residues within the helix α3 of DNA binding domains (DBDs) contribute to constitutive or inducible nuclear import for all zebrafish IRF family members and DNA binding for most members, thereby enabling them to function as transcription factors. Our results reveal a conserved and general mechanism that specifies zebrafish IRF family proteins to nuclear import and DNA binding, thereby regulating fish IFN response.

STK39 is an independent risk factor for male hypertension in Han Chinese.

BACKGROUND: STK39 interacts with OXSR1 and phosphorylates the sodium-chloride co-transporter (SLC12A3), which plays a critical role in regulating the salt/water balance and blood pressure. Here we tested whether STK39, OXSR1, and SLC12A3 genetically contribute to hypertension in the Han Chinese population and how the SNP to SNP or SNP to other risk factors interacts in the pathogenesis of hypertension. METHODS AND RESULTS: Eleven tagging SNPs from STK39, OXSR1, and SLC12A3 were selected and first genotyped in 1210 hypertensive and healthy individuals by sequencing. Two SNPs of STK39, rs6433027 and rs3754777, were found to be associated with hypertension in males (P=0.008-0.024). All other SNPs were not associated with hypertension in either gender. The association of rs6433027 and rs3754777 with male hypertension was validated by genotyping another 4598 hypertensive and healthy individuals. The odds ratios (95% confidence interval, P value) in males were 1.269 (1.13-1.43; P=0.0001) and 1.231 (1.078-1.41; P=0.004) of rs6433027 and rs3754777, respectively. The allele T of rs6433027 presented a strong epistatic effect on the allele A of rs3754777 in hypertensive trait. The minor allele frequencies of two SNPs were not stratified by age, BMI, or diabetes, the three major risk factors for hypertension. CONCLUSION: Our results suggest that STK39 is an independent risk factor for hypertension in men and that its intragenic SNPs can interact and function in the control of blood pressure.

Bis{(E)-4-bromo-2-[(2-chloro-3-pyrid-yl)imino-meth-yl]phenolato-κN,O}copper(II).

In the title complex, [Cu(C(12)H(7)BrClN(2)O)(2)], the Cu(II) center is tetra-coordinated by two phenolate O and two azomethine N atoms from two independent bidentate 4-bromo-2-[(2-chloro-3-pyrid-yl)imino-meth-yl]phenolate (L) ligands. In the crystal structure, the Cu(II) atom has a distorted square-planar coordination environment. The inter-planar dihedral angles between the benzene and pyridine rings in the individual ligands are 63.83 (4) and 54.43 (3)°, indicating the pyridine ring to have considerably weaker steric hindrance.

Vaccine with beta-defensin 2-transduced leukemic cells activates innate and adaptive immunity to elicit potent antileukemia responses.

Murine beta-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-gamma production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL.

LL-37 enhances adaptive antitumor immune response in a murine model when genetically fused with M-CSFR (J6-1) DNA vaccine.

DNA vaccine against M-CSFR(J6-1) (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFR(J6-1) DNA vaccines, we constructed genetically fused vaccines encoding M-CSFR(J6-1) and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFR(J6-1)), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells, and inducing IFN-gamma and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells. Our results suggest that when genetically fused with M-CSFR(J6-1), LL-37 could enhance adaptive immune response against M-CSFR(J6-1) in a murine model challenged with tumor cells bearing M-CSFR(J6-1).

Marked reduction of LL-37/hCAP-18, an antimicrobial peptide, in patients with acute myeloid leukemia.

We detected LL-37/hCAP-18 expression in the peripheral blood smears of 50 healthy donors and 143 patients with various hematological diseases. Compared with that in the healthy donors, expression of the protein in the neutrophils was significantly lower in patients with acute myeloid leukemia (AML), especially those with infection, but no significant difference was detected in messenger RNA level. We did not detect increased LL-37/hCAP-18 protein expression in U937 cells treated with lipopolysaccharide or Staphylococcus aureus Cowan strain. Furthermore, LL-37/hCAP-18 protein production was not restored in differentiated myeloid cell lines NB4 or HL-60 induced by all-trans retinoic acid. LL-37/hCAP-18 has been shown to play a role in host defense, and its deficiency in AML may be one of the explanations for susceptibility to infection among these patients.

[Expression of gelatinase A and B in cord blood CD34+ cells and leukemic cell lines].

OBJECTIVE: To study the expression of gelatinases, including matrix metalloproteinase-9 (MMP-9, gelatinase B) and matrix metalloproteinase-2 (MMP-2, gelatinase A), in CD(34)(+) cells and leukemic cell lines, and explore the significance of gelatinase in migrating and homing capacity of CD(34)(+) cells, as well as the role of gelatinase in leukemia pathogenesis. METHODS: CD(34)(+) cells were isolated from umbilical cord blood and normal bone marrow by Mini MACS system. By zymogram analysis, MMP-2 and MMP-9 were detected in the serum free condition medium of CD(34)(+) cells and cell lines. RESULTS: One brilliant band with molecular weight of 92 x 10(3) was detected in condition medium of cord blood CD(34)(+) cells. No band was detected in condition medium of bone marrow CD(34)(+) cells. Brilliant bands with molecular weight of 92 x 10(3) and 72 x 10(3) were detected in the condition medium of U937, KG-1a and HL-60 cell lines, but not in that of HEL, Namalva, CEM, K562 and LCL-H cell lines. In the condition media of J6-1 and J6-2 cells only the 92 x 10(3) band was detected. CONCLUSIONS: Cord blood CD(34)(+) cells produced MMP-9, but bone marrow CD(34)(+) cells did not, partly explains the fact that cord blood CD(34)(+) cells possessed higher migrating capacity in comparison with bone marrow CD(34)(+) cells. The expression of MMP-9 and MMP-2 in leukemic cell lines varied.